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Retrograde impulse activity and horseradish peroxidase tracig of nerve fibers entering neuroma studied in vitro
EXPERIMENTAL
NEUROLOGY
85.400-4
12 ( 1984)
Retrograde Impulse Activity and Horseradish Peroxidase Tracing of Nerve Fibers Entering Neuroma Studied in Vitro J. D. KOCSIS, R. J. PRESTON, AND E. F. TARG’ Department of Neurology, Stanford University School of Medicine, and Palo Alto Veterans Administration Medical Center, Palo Alto, California 94307 Received
March
15, 1984
Neuroma formation was induced in rat sciatic nerve by tight ligature. At various times after placement of the ligature, the nerves were excised, desheathed, split longitudinally proximal to the neuroma, and studied in vitro in a nerve chamber. Stimulation of one of the arbitrarily formed proximal branches was found to generate impulse activity in the other branch. Since similar branch to branch activation did not occur in control preparations, it appeared that some form of axon to axon interaction occurred within the neuroma, or alternatively that retrograde regeneration allowed continuity of nerve fibers in proximal divisions of the nerve trunk. Attempts at morphological demonstration of the continuity alternative were made by applying horseradish peroxidase to cut fibers of one proximal division. Although labeled axons did turn retrogradely within the neuroma, they were not found to enter the other nerve division.
INTRODUCTION Mammalian myelinated nerve fibers regenerate in a relatively orderly manner following a discrete nerve crush ( 17, 2 1). The axon cylinder is disrupted but the basement membrane of the Schwann cell remains intact to provide a “channel” for the regenerating axon (21). However, after tight ligature or procedures that prevent fibers from reestablishing peripheral connections, regeneration occurs at the end of the nerve to form a bulbous enlargement, or neuroma (2, 17,2 1). A number of abnormal electrophysiologic properties have been attributed to fibers within a neuroma (3, $6, 12, 13, 19,20, 23). ’ This work was supported in part by grants from the National Institute of Health and the Kroc Foundation; and by the Medical Research Service of the Veterans Administration, and the Neural Plasticity Program of Stanford University Medical School. E. F. Targ was supported in part by the Medical Scholars Program at Stanford University Medical School. 400 0014-4886/84 $3.00 Copyri&t 0 1984 by Academic Press. Inc. All r’i&ts of tqmduction in any form reserved.
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One of these (also a property of dysmyelinated axons of the dystrophic mouse (7, 18)) is the apparent ability for impulses in some axons to propagate into the region of pathology and activate neighboring fibers (3, 13,20). In normal nerve, impulse activity in one set of axons has a weak effect on excitability of neighboring axons, and rarely produces spike activity in them (1, 8, 9, 15). Considerable attention has focused on the study of such cross-talk between nerve fibers that enter neuroma because of the possibility that it may contribute to abnormal pain and paresthesia in patients with peripheral nerve injuries. Cross-talk has been linked to the occurrence of abnormal retrograde activity that follows normal anterograde propagation into the neuroma (3, 13, 20). But, Blumberg and Janig (3) have proposed that not all of this retrogrde activity is the result of cross-talk. They suggest that retrograde regeneration may occur and account for some of the retrograde impulses. We characterized retrograde impulse activity in an in vitro neuroma preparation, and then used horseradish peroxidase (HRP) to determine whether or not retrograde regeneration could be shown to provide a substrate for the activity. Our results suggested that retrograde regeneration may not underly all retrograde activity observed in this preparation. METHODS The sciatic nerves of anesthetized (sodium pentobarbital; 50 mg/kg) Wistar rats were exposed and a tight ligature was made around them low in the thigh. The nerves were cut distal to the tie. At various recovery times (3 weeks to 13 months) the animals were again anesthetized, exsanguinated, and their sciatic nerves removed. The nerves were placed in normal Ringer’s solution (in mM: NaCl, 124; KCl, 3.0; MgS04, 2.0; CaC12, 2.0; NaHC03, 26.0; and dextrose, 10.0; pH, 7.4, equilibrated with 95% 02-5% CO;?). After removal of the epineurial sheath, it was seen that several branches of the sciatic nerve were included in the ligature and that each one formed a separate bulbous enlargement. The largest branch with its bulbous head was selected for study. The nerve segment proximal to the neuroma ( 1.5 cm) was transected longitudinally with iridectomy scissors. The split nerve preparations were then placed in a nerve recording chamber. The neuroma head was continuously superfused with Ringer’s solution. Stimulating and recording electrodes were positioned on both divisions of the nerve as shown in Fig. 2. Nerve recording and stimulating methods were conventional and are described more fully in Kocsis et al. (11). In eight experiments, HRP procedures described by Preston et al. (16) were used to look for axon continuity between proximal divisions of the nerve trunk. In these procedures one proximal division was trimmed to a length of 2.8 to 5 mm and its cut end was exposed 2 h to HRP (3% in
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Ringer’s solution at 5°C). The preparation was then rinsed and stored 28 to 54 h in Ringer’s (5°C) prior to glutaraldehyde fixation. The storage period was selected to accord with the estimated length of fiber trajectories through the preparation and with a previously estimated HRP diffusion rate of 0.25 mm/h in medium to large diameter rat sciatic axons. After fixation and bu!Ter rinse the preparation was frozen whole and then thawed or was sectioned with a freezing microtome prior to reaction with 3,3’diaminobenzidine tetrahydrochloride (DAB, 0.05%) and Hz02 (0.0024%). The distribution of HRPlabeled axons was determined light microscopically after embedding the samples in Permount or Vestopal or after glycerol dehydration. RESULTS In normal nerves weak electrical field effects from surround impulse activity can lead to changes in the excitability of neighboring nonactivated fibers but these influences do not appear to lead to spike initiation (9). In control preparations with normal nerve we established the absence of spike-inducing held effects by using the schema of Fig. 1A. This preparation involved the arrangement of stimulating and recording electrodes on a normal sciatic nerve that had been divided longitudinally to within 2 mm of its distal end. The lengths of the divisions varied from 8 to 12 mm. A discrete compound action potential could be recorded along the same nerve division that was stimulated (Fig. 1B). If the impulses that entered the joined nerve trunk after stimulation of this branch (site 1) led to excitation of neighboring axons, activity should be detected in the other branch (site 3). No activity could be recorded from the other branch, thereby indicating that impulse activity from one division of the normal nerve did not generate activity in nearby axons that originated from the other division. Six normal nerves were studied in this manner and none showed evidence for branch to branch activity. Similar experiments had a different outcome when the nerves terminated distally in a neuroma as illustrated in Fig. 2A. Again as expected, relatively discrete compound action potentials were registered at electrodes on the stimulated proximal division (Fig. 2B). This response was typically more complex in the neuroma preparation (i.e., late, low-amplitude activity after an initial negative wave) than in normal nerve (Fig. 1) where the discrete compound response was completed within 1.5 ms after the stimulus. The same stimuli produced responses at the neck of the neuroma (Fig. 2C; from site 3), where an initial large-amplitude negative wave was still distinct from later, irregular activity, and in the head of the neuroma (Fig. 2D, from site 2) where early and late activity appeared to fuse into a long-duration complex wave. Furthermore, and in marked contrast to control preparations, stimulation of one proximal nerve division also produced responses in the non-
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FIG. 1. A-schematic of stimulating and recording electrode arrangement for normal nerve split longitudinally. B-whole-nerve recording obtained at site 2 after stimulation at site I. Cno impulse activity could be recorded at site 3 after site 1 stimulation in normal nerve. Positive voltage deflections are up in this and subsequent figures.
activated proximal division (Fig. 2E, F). This branch to branch response (hereafter the transneuroma response) consisted of several dispersed components having variable amplitudes. For example, the earliest activity in the response of Fig. 2E and F began with a latency of 1.1 ms but this was followed by low-amplitude negative waves for nearly 12 ms. Both the transneuroma response and some portion of the responses recorded from the stimulated nerve division were affected by crush near the neuroma (dashed line in Fig. 2A). The transneuroma response (and the response recorded from the head of the neuroma) was completely abolished (Fig. 2H) by the crush, and the response recorded from the stimulated nerve division was modified in shape. This latter effect is illustrated in Fig. 21 and J where it is seen that the crush manipulation removed the irregular late activity (25) and narrowed the initial negative wave (21) (compared with the precrush response of Fig. 2B).
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FIG. 2. A-schematic of recording and stimulating electrode arrangement of a nerve split longitudinally that enters a neuroma head. The lower branch was stimulated and recordings were obtained on the same branch (site 1), the head (site 2), or neck (site 3) of the neuroma, or from the other branch (site 4). Recordings were obtained from site 1 on the stimulated branch (B), and from sites 3 (C) and 2 (D). Note the irregularities and late activity in the three responses (arrow in B). The response recorded from one branch (site 4) after stimulation of the other branch (the transneuroma response) is shown in E and at a higher oscilloscope gain in F. Gextracellular unit responses recorded from the neuroma head aher stimulation of one branch (first arrow) and another branch (second arrow). H-after crush at the neck (dashed line in A), responses from the head (upper trace) and the other branch (lower trace) were obliterated. Irecordings obtained from site 1 had a conspicuous loss of late activity after the crush. The highergain traces in J show the late activity before (arrow) and after crush. The voltage calibration in B pertains to B-E, and H-J; those of F and G refer to those traces. The time calibration in I is for all traces.
Transneuroma responses could be obtained from either proximal nerve division after stimulation of the other division and were found in 34 preparations with postligature times ranging from 3 weeks to 13 months. In addition to the demonstration of transneuroma activity with compound action potentials, some extracellular unit correlates of this phenomenon were obtained with microelectrode penetrations into the neuroma and proximal nerve divisions. Figure 2G shows extracellularly recorded spikes of a fiber in the head of the neuroma after stimulation of either proximal nerve branch. It was interesting to note the large latency difference for these responses despite the equality of conduction distances measured macroscopically to stimulation sites in the proximal nerves.
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Eight nerve segments from which transneuroma responses had been recorded were used for HRP study. These experiments were carried out to look for axon continuity between proximal nerve divisions and are represented by the schematic of Fig. 3A. The enzyme was applied to the cut end of one proximal division (D-A) which was previously trimmed to a length (X) of 2.8 to 5.0 mm. The nondivided distal portion of the neuroma had a length (Y) of 1.5 to 5.0 mm (1.5 to 2.2 mm in four samples). Because retrograde looping was anticipated to begin at the distal end of the neuroma, it was expected that labeled axons would have to be followed over a distance of X + 2Y. This distance ranged from 7.0 to 12.8 mm (mean 9.9 mm). In the HRP-dipped nerve division, the reaction product inside fine-, medium-, and large-diameter axons had a Go&like appearance comparable to that known for central nervous system axons to follow “injury uptake” of
FIG. 3. HRP-labeled axons in neuroma after application of enzyme to a portion of the proximal nerve. A-schematic representation of preparation and resultant distribution of labeled axons (see text). B-D--50~pm-thick sections from one preparation. In B, labeled axons entered the neuroma from the soaked proximal nerve division but did not spread throughout the neuroma and did not enter nonsoaked proximal nerve. Retrograde turns of tibers at arrows are enlarged in D. In C, many fibers that reach the distal end of the neuroma (another section) turned and continued a retrograde course among labeled antergrade segments. In D, two fibers made sharp retrograde turns. Other reaction product was in more faintly labeled axons as well as in short segments of blood vessels.Magnifications are X25 in B, X63 in C and X 100 in D.
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HRP. Labeled axons were scattered throughout this division and extended into the distal, nondivided stump, but they were always more numerous in the former area than in the latter (Figs. 3B, 4A). In every preparation there was a well defined and cohesive area of the neuroma that contained virtually no labeled axons (stippled area NHB in Fig. 3A; also Fig. 3B). The fibers in this area of the neuroma appeared to be distal extensions of fibers from the proximal nerve division that was not exposed to HRP (D-B). Reaction product
nc. 4. HRP-labeled axons in a whole-mount preparation. A-labeled axons extended from the proximal nerve segment that is twisted diagonally at the upper left. B-enlarged portion of the neuroma showing groups of fibers near the surface of the bundle that made retrograde turns. Magnifications are x47 in A and X150 in B.
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was never found in axons of segment D-B, nor were any examples found of labeled processes directing their course toward area NH-B in a manner suggesting that they would have continued a course into segment D-B. Because the preceeding observations were made in seven whole-mount samples, it was important to consider that reaction product was not formed throughout the tissue mass. This was determined in control nerves that were sectioned after DAB processing as whole segments. These controls revealed densely labeled fibers near the periphery of the bundle, gradual lessening of reaction product density in fibers situated more centrally in the nerve, and absence of reaction product in fibers at distances greater than 200 to 250 pm from the surface. It must be concluded that DAB was not able to react with all HRP present in the whole-nerve samples. However, the pattern of HRP labeling described above for whole-nerve experimental material was also obtained in each of eight 50-pm-thick sections from one neuroma sample (frozen sections taken before reaction), and in this case the probability of undetected HRP was greatly reduced. Among all labeled fibers that coursed within the neuroma, relatively few axons erratically changed direction during any portion of their observed path. Most fibers followed courses that were parallel to the longitudinal axis of the preparation. Frequently the reaction product in a fiber faded gradually and became undetectable, sometimes ending at the distal tip of the neuroma but more often disappearing at intermediate levels of the neuroma. Other labeled fibers made single loops (Figs. 3C, D, 4B), often with an extremely small radius, and continued a retrograde course parallel to and at times slowly rotating around the anterograde segment (Fig. 3D). Such loops were more prominent at the distal end of the neuroma but they could be found at more proximal levels. Except in one preparation (with Y length of 1.5 mm) retrogradely directed segments from loops in the distal extreme of the neuroma could not be readily traced to the level of the nerve division. However, there was little tendency for fibers to direct their retrograde extensions toward the nonlabeled area of the neuroma. Some labeled axons issued collateral branches at distances of 1.5 mm or more from the distal tip of the neuroma. Those collaterals most often took an anterograde direction and generally coursed near the parent axon. DISCUSSION Peripheral nerve fibers that enter a neuroma show a number of abnormal electrophysiologic properties. These include increased mechanosensitivity, spontaneous firing, and afterdischarges (3, 5, 6, 12, 13, 19, 20, 23). Another property attributed to neuroma but not to normal nerve is a high safety factor axonal “cross-talk” (3, 13, 20). This phenomenon was originally proposed to explain experiments where a neuroma existed in sciatic nerve and
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where stimulation of a dorsal root that was cut centrally led to activation of ventral root fibers (20). Although the mechanism of this effect is uncertain, it has been assumed that morphologic alterations within the neuroma such as close apposition of the fine-caliber axonal sprouts could allow for electrical coupling between the axons (2). No contact specializations such as gap junctions or synaptic-like profiles have been described for fibers entering neuroma, and recent studies have raised the prospect that retrograde regeneration from a neuroma may at least partially account for retrograde impulse activity (3). One aim of the present work was to establish whether or not some physiologic properties associated with peripheral neuroma studied in viva could also be shown with a simple in vitro model. It was found that abnormal responses were reliably elicited in the in vitro preparation. The most conspicuous abnormality occurred when stimulation of one arbitrarily formed proximal division of a nerve resulted in transneuroma activity in fibers of another, nonstimulated, division. There are several arguments against the possibility that this activity resulted from inadvertent stimulus currents acting on tissues remote from the stimulation electrodes (i.e., virtual cathode effects). Firstly, the transneuroma response occurred with a relatively long latency whereas electrotonic spread of stimulus current could be expected to produce short-latency responses. Secondly, the transneuroma response was abolished by crush at the neck of the neuroma. And finally, similar responses could not be elicited in simulations of the neuroma experiments with split segments ending as nondivided normal nerve. Although our electrophysiologic data do not entirely establish the morphologic basis for transneuroma responses, several conclusions can be drawn. First, the very occurrence of these responses in the split-neuroma preparation confirms that at least some portion of the transneuroma response must be due either to direct axonal connection (via retrograde regeneration) between proximal nerve divisions or to axonal cross-talk in the head of the neuroma, or to some combination of these factors. However, if a single stimulus gives rise to repetitive firing, parts of the early and late components of the transneuroma response may originate from the same fiber. The late activity in the response of the stimulated branch may share a common mechanism to the transneuroma response, i.e., resulting from retrograde regeneration or cross-talk. There is the additional possibility of impulse reflection back into the stimulated branch due to morphologic nonhomogeneity of the axon region within the neuroma (22). We have not been able to distinguish between these three possibilities from our electrophysiological studies. However, we would like to point out that if axons entering neuroma regenerate in a retrograde manner and loop so that stimulation of a given whole-nerve branch leads to activation of both ends of the looping axon, then under ideal conditions, collision should occur and the late activity as recorded from the
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stimulated branch should be obliterated. Similarly, if cross-talk occurs within the neuroma, collision should also occur after stimulation of a given branch with attendant loss of late activity. However, late activity in the stimulated branch in our experiments was prominent. Yet, the highly nonhomogeneous morphologic organization of the axons as they enter the neuroma could significantly alter the safety factor for transmission and could explain the lack of collision. Specifically, the conduction safety factor could be lower for one branch thereby leading to impulse failure in or near the neuroma. If failure occurs along the propagation path of one arm of the looping axon (or one element of a coupled pair of axons), the other activated axon may (given a high enough safety factor) continue retrograde propagation and give rise to activity in the stimulated branch. A second aim of this work was to show, with HRP diffusion, whether or not axons would course toward the neuroma from one division of the proximal nerve and then proceed into the other proximal division. Our failure to demonstrate this direct connectivity is offered only as tentative morphologic evidence that axonal cross-talk may be the determinant of the transneuroma response. Although no HRP was found in the nonsoaked nerve division of eight samples, the following criticisms can be made: (i) The observed transneuroma responses may have been generated by retrograde activity in a relatively small set of axons. In the majority of eight samples, relatively few retrograde segments could be traced to and beyond the region of nerve division. Thus, it is possible that HRP did not diffuse far enough, in the relevant set of axons, to enter the nonsoaked division. (ii) In seven of eight samples (whole-tissue specimens) as much as 40% of the core tissue volume that contained HRP probably did not form reaction product. Thus a small set of retrograde loops contained within this volume in the nonsoaked division would not have formed reaction product despite possibly containing HRP. (iii) The HRP may not have diffused into all axons of the soaked division and therefore a small set of fibers that ultimately coursed into the nonsoaked division may have been consistently excluded from the set of labeled axons. It should be emphasized that the latter two possibilities would have acted randomly and therefore seem improbable causes of failure to detect a single positive outcome in eight samples. Furthermore, several features of our data should be given appropriate weight: (a) Retrograde extensions were traced to the region of nerve division thereby indicating that diffusion distance of HRP was sufficient in a set of axons. (b) Retrograde extensions originated from loops in all regions of the neuroma (not just from those in the distal termination), further increasing the likelihood of tracing labeled axons into the nondipped branch. (c) In all HRP experiments, retrograde segments rarely
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coursed into any part of the nonlabeled mass of the neuroma. (d) There was a marked tendency for a fiber’s retrograde segment to course very close to its anterograde segment, suggesting that both segments would most likely be found in the same proximal division of the nerve. Taken together, these observations lend support to the hypothesis that retrograde transneuroma activity in these experiments may have been caused by cross-talk rather than by direct continuity of axons between the divisions of the proximal nerve. Other investigators have suggested that “ephaptic” (electrical field effect) interactions account for this activity (2, 3, 5, 13, 20, 23). However, there is no direct confirmation of this proposal. Several morphologic features of axons entering neuroma formation offer support for this proposal. The fibers that enter neuroma give rise to fine-caliber axonal sprouts that can become closely apposed with few intervening glial elements (2). The reasoning to explain why densely packed axonal sprouts are more likely to give rise to electrical interactions is that the extracellular resistance is increased from the tight axon packing and more current will be shunted across neighboring axon membranes ( l&20). As few glial elements intervene, there are few alternative current pathways except through the extracellular space and the neighboring axons. In the cerebellar cortex a similar morphologic organization is present, i.e., dense, fine-caliber axon packing with few intervening glial elements. Activity-dependent changes in extracellular potassium can lead to profound changes in axonal excitability of nonactivated nonmyelinated axons (10). Whereas the excitability changes brought about from change in the extracellular ionic environment can last for seconds, cross-talk from electrical field effects more closely approximates the time course of the action potential (9). As pointed out by Seltzer and Devor (20), the high safety coupling between nerve fibers in neuroma is not likely due to increase in extracellular potassium because its time course is brief and the effect is nonaccumulative with repetitive stimulation. Analytical studies of interactions of nonmyelinated fibers (4, 14) and arguments set forth by Rasminsky (18) indicate that myelinated fibers may be more susceptible than nonmyelinated axons to electrical field effect excitation from surround impulse activity. This may be because myelinated fibers have a relatively low membrane capacitance and low axial resistance, both of which lead to an increased probability of interaction. Indeed, Rasminsky (18) demonstrated in dysmyelinated fibers of the dystropic mouse that interactions more readily proceed from unmyelinated to myelinated axons. Previous studies on axonal interactions or cross-talk in neuroma have been carried out in Go. In this study we demonstrated that nerves terminating
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in neuroma can be maintained in vitro and that a transneuroma response can be demonstrated in this relatively simple in vitro preparation. An advantage of this preparation is that alteration in the ionic environment and direct application of drugs to the neuroma can be made. The technique should prove valuable in future studies to determine in more detail the specific mechanisms responsible for transneuroma activity and for other abnormal activity of pathological fibers entering the neuroma. REFERENCES 1. ARVANITAKI, A. 1942. Effects evoked in an axon by activity of a contiguous one. J. Neurophysiol. 5: 89-108. 2. BERNSTEIN, J. J.. AND D. PAGNANELLI. 1982. Long-term axonal apposition in rat sciatic nerve neuroma. J. Neurosurg. 57: 682-684. 3. BLUMBERG, H., AND W. JANIG. 1982. Activation of fibers via experimentally produced stump neuromas of skin nerves: ephaptic transmission or retrograde sprouting. Exp. Neural.
16: 468-482.
4. CLARK, J. W., AND R. PLONSEY. 1970. A mathematical study of nerve fiber interaction. Biophys.
J. 10: 937-957.
5. DEVOR, M., AND J. J. BERNSTEIN. 1982. Abnormal impulse generation in neuromas: electrophysiology and ultrastructure. Pages 363-80 in J. OCHOA AND B. CULP, eds., Abnormal Nerves and Muscles as Impulse Generators. Oxford Univ. Press, London/New York. 6. GROVRIN-LIPPMANN, R., AND M. DEVOR. 1978. Ongoing activity in severed nerves: source and variation with time. Brain Res. 159: 406-410. 7. HUIZAR, P., M. KUNO, AND Y. MIYATA. 1975. Electrophysiological properties of spinal motoneurones of normal and dystrophic mice. J. Physiol. (London) 248: 231-246. 8. KATZ, B., AND 0. H. SCHMITT. 1942. A note on interaction between nerve fibers. J. Physiol. (London) 100: 369-37 1. 9. KOCSIS, J. D., J. A. RUIZ, AND K. CUMMINS. 1982. Modulation of axonal excitability mediated by surround electrical activity; an intra-axonal study. Exp. Brain Res. 47: 15 l153. 10. Kccsn, J. D., R. C. MALENKA, AND S. G. WAXMAN. 1983. Effectsof extracellular potassium on the excitability of the parallel fibers of the cerebellar cortex. J. Physiol. (London) 334: 225-244.
1I. KOCSIS,J. D., J. A. RUIZ, AND S. G. WAXMAN. 1983. Maturation of mammalian myelinated fibers: changes in action potential characterization following 4-aminopyridine application. J. Neurophysiol.
50: 449-463.
12. KORENMAN, E. M. D., AND M. DEVOR. 198 I. Ectopic adrenergic sensitivity in damaged peripheral nerve axons in the rat. Exp. Neural. 72: 63-81. 13. LISNEY, S. J. W., AND C. M. POVER. 1983. Coupling between fibres involved in sensory nerve neuroma in cats. J. Neural. Sci. 59: 255-264. 14. MAEDA, K.. T. YAGI, AND A. NOCXJCHI. 1980. Induced excitation and synchronization of nerve impulses in two parallel unmyelinated fibers. IEEE Trans. Biomed. Eng. 27: 139145. 15. MARRAZZI, A. S., AND R. LORENTE DE N6. 1944. Interaction of neighboring fibers in myelinated nerve. J. Neurophysiol. 7: 83-101. 16. PRESTON,R. J., J. D. KOCSIS, AND S. G. WAXMAN. 1983. Axon-myelin anomolies revealed
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KOCSIS, PRESTON, AND TARG by diaminobenzidine reaction with intra-axonal horseradish peroxidase. Sot. Neurosci. Abstr. 9: 50. kUdN Y CAJAL, S. 1928. Degeneration and Regeneration of the Nervous System. Vol. 1. Oxford Univ. Press, London. RASMINSKY, M. 1980. Ephaptic transmission between single nerve fibres in the spinal nerve roots of dystrophic mice. J. Physior (London) 305: 15 l- 169. SCADDING,J. W. 198 1. Development of ongoing activity, mechanosensitivity, and adrenalin sensitivity in severed peripheral axons. Exp. Neural. 73: 345-364. SELTZER,Z., AND M. DEVOR. 1979. Ephaptic transmission in chronically damaged peripheral nerves. Neurology 29: 106 I-1064. SUNDERLAND, A. 1968. Nerves and Nerve Injuries. Livingston, Edinburgh. SWADLOW,H. A., J. D. Kocs~s, AND S. G. WAXMAN. 1980. Modulation of impulse conduction along the axonal tree. Annu. Rev. Biophys. Bioeng. 9: 143-79. WALL, P., AND M. GUTNICK. 1974. Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Exp. Neural. 43: 580-593.
NEUROLOGY
85.400-4
12 ( 1984)
Retrograde Impulse Activity and Horseradish Peroxidase Tracing of Nerve Fibers Entering Neuroma Studied in Vitro J. D. KOCSIS, R. J. PRESTON, AND E. F. TARG’ Department of Neurology, Stanford University School of Medicine, and Palo Alto Veterans Administration Medical Center, Palo Alto, California 94307 Received
March
15, 1984
Neuroma formation was induced in rat sciatic nerve by tight ligature. At various times after placement of the ligature, the nerves were excised, desheathed, split longitudinally proximal to the neuroma, and studied in vitro in a nerve chamber. Stimulation of one of the arbitrarily formed proximal branches was found to generate impulse activity in the other branch. Since similar branch to branch activation did not occur in control preparations, it appeared that some form of axon to axon interaction occurred within the neuroma, or alternatively that retrograde regeneration allowed continuity of nerve fibers in proximal divisions of the nerve trunk. Attempts at morphological demonstration of the continuity alternative were made by applying horseradish peroxidase to cut fibers of one proximal division. Although labeled axons did turn retrogradely within the neuroma, they were not found to enter the other nerve division.
INTRODUCTION Mammalian myelinated nerve fibers regenerate in a relatively orderly manner following a discrete nerve crush ( 17, 2 1). The axon cylinder is disrupted but the basement membrane of the Schwann cell remains intact to provide a “channel” for the regenerating axon (21). However, after tight ligature or procedures that prevent fibers from reestablishing peripheral connections, regeneration occurs at the end of the nerve to form a bulbous enlargement, or neuroma (2, 17,2 1). A number of abnormal electrophysiologic properties have been attributed to fibers within a neuroma (3, $6, 12, 13, 19,20, 23). ’ This work was supported in part by grants from the National Institute of Health and the Kroc Foundation; and by the Medical Research Service of the Veterans Administration, and the Neural Plasticity Program of Stanford University Medical School. E. F. Targ was supported in part by the Medical Scholars Program at Stanford University Medical School. 400 0014-4886/84 $3.00 Copyri&t 0 1984 by Academic Press. Inc. All r’i&ts of tqmduction in any form reserved.
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One of these (also a property of dysmyelinated axons of the dystrophic mouse (7, 18)) is the apparent ability for impulses in some axons to propagate into the region of pathology and activate neighboring fibers (3, 13,20). In normal nerve, impulse activity in one set of axons has a weak effect on excitability of neighboring axons, and rarely produces spike activity in them (1, 8, 9, 15). Considerable attention has focused on the study of such cross-talk between nerve fibers that enter neuroma because of the possibility that it may contribute to abnormal pain and paresthesia in patients with peripheral nerve injuries. Cross-talk has been linked to the occurrence of abnormal retrograde activity that follows normal anterograde propagation into the neuroma (3, 13, 20). But, Blumberg and Janig (3) have proposed that not all of this retrogrde activity is the result of cross-talk. They suggest that retrograde regeneration may occur and account for some of the retrograde impulses. We characterized retrograde impulse activity in an in vitro neuroma preparation, and then used horseradish peroxidase (HRP) to determine whether or not retrograde regeneration could be shown to provide a substrate for the activity. Our results suggested that retrograde regeneration may not underly all retrograde activity observed in this preparation. METHODS The sciatic nerves of anesthetized (sodium pentobarbital; 50 mg/kg) Wistar rats were exposed and a tight ligature was made around them low in the thigh. The nerves were cut distal to the tie. At various recovery times (3 weeks to 13 months) the animals were again anesthetized, exsanguinated, and their sciatic nerves removed. The nerves were placed in normal Ringer’s solution (in mM: NaCl, 124; KCl, 3.0; MgS04, 2.0; CaC12, 2.0; NaHC03, 26.0; and dextrose, 10.0; pH, 7.4, equilibrated with 95% 02-5% CO;?). After removal of the epineurial sheath, it was seen that several branches of the sciatic nerve were included in the ligature and that each one formed a separate bulbous enlargement. The largest branch with its bulbous head was selected for study. The nerve segment proximal to the neuroma ( 1.5 cm) was transected longitudinally with iridectomy scissors. The split nerve preparations were then placed in a nerve recording chamber. The neuroma head was continuously superfused with Ringer’s solution. Stimulating and recording electrodes were positioned on both divisions of the nerve as shown in Fig. 2. Nerve recording and stimulating methods were conventional and are described more fully in Kocsis et al. (11). In eight experiments, HRP procedures described by Preston et al. (16) were used to look for axon continuity between proximal divisions of the nerve trunk. In these procedures one proximal division was trimmed to a length of 2.8 to 5 mm and its cut end was exposed 2 h to HRP (3% in
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Ringer’s solution at 5°C). The preparation was then rinsed and stored 28 to 54 h in Ringer’s (5°C) prior to glutaraldehyde fixation. The storage period was selected to accord with the estimated length of fiber trajectories through the preparation and with a previously estimated HRP diffusion rate of 0.25 mm/h in medium to large diameter rat sciatic axons. After fixation and bu!Ter rinse the preparation was frozen whole and then thawed or was sectioned with a freezing microtome prior to reaction with 3,3’diaminobenzidine tetrahydrochloride (DAB, 0.05%) and Hz02 (0.0024%). The distribution of HRPlabeled axons was determined light microscopically after embedding the samples in Permount or Vestopal or after glycerol dehydration. RESULTS In normal nerves weak electrical field effects from surround impulse activity can lead to changes in the excitability of neighboring nonactivated fibers but these influences do not appear to lead to spike initiation (9). In control preparations with normal nerve we established the absence of spike-inducing held effects by using the schema of Fig. 1A. This preparation involved the arrangement of stimulating and recording electrodes on a normal sciatic nerve that had been divided longitudinally to within 2 mm of its distal end. The lengths of the divisions varied from 8 to 12 mm. A discrete compound action potential could be recorded along the same nerve division that was stimulated (Fig. 1B). If the impulses that entered the joined nerve trunk after stimulation of this branch (site 1) led to excitation of neighboring axons, activity should be detected in the other branch (site 3). No activity could be recorded from the other branch, thereby indicating that impulse activity from one division of the normal nerve did not generate activity in nearby axons that originated from the other division. Six normal nerves were studied in this manner and none showed evidence for branch to branch activity. Similar experiments had a different outcome when the nerves terminated distally in a neuroma as illustrated in Fig. 2A. Again as expected, relatively discrete compound action potentials were registered at electrodes on the stimulated proximal division (Fig. 2B). This response was typically more complex in the neuroma preparation (i.e., late, low-amplitude activity after an initial negative wave) than in normal nerve (Fig. 1) where the discrete compound response was completed within 1.5 ms after the stimulus. The same stimuli produced responses at the neck of the neuroma (Fig. 2C; from site 3), where an initial large-amplitude negative wave was still distinct from later, irregular activity, and in the head of the neuroma (Fig. 2D, from site 2) where early and late activity appeared to fuse into a long-duration complex wave. Furthermore, and in marked contrast to control preparations, stimulation of one proximal nerve division also produced responses in the non-
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FIG. 1. A-schematic of stimulating and recording electrode arrangement for normal nerve split longitudinally. B-whole-nerve recording obtained at site 2 after stimulation at site I. Cno impulse activity could be recorded at site 3 after site 1 stimulation in normal nerve. Positive voltage deflections are up in this and subsequent figures.
activated proximal division (Fig. 2E, F). This branch to branch response (hereafter the transneuroma response) consisted of several dispersed components having variable amplitudes. For example, the earliest activity in the response of Fig. 2E and F began with a latency of 1.1 ms but this was followed by low-amplitude negative waves for nearly 12 ms. Both the transneuroma response and some portion of the responses recorded from the stimulated nerve division were affected by crush near the neuroma (dashed line in Fig. 2A). The transneuroma response (and the response recorded from the head of the neuroma) was completely abolished (Fig. 2H) by the crush, and the response recorded from the stimulated nerve division was modified in shape. This latter effect is illustrated in Fig. 21 and J where it is seen that the crush manipulation removed the irregular late activity (25) and narrowed the initial negative wave (21) (compared with the precrush response of Fig. 2B).
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FIG. 2. A-schematic of recording and stimulating electrode arrangement of a nerve split longitudinally that enters a neuroma head. The lower branch was stimulated and recordings were obtained on the same branch (site 1), the head (site 2), or neck (site 3) of the neuroma, or from the other branch (site 4). Recordings were obtained from site 1 on the stimulated branch (B), and from sites 3 (C) and 2 (D). Note the irregularities and late activity in the three responses (arrow in B). The response recorded from one branch (site 4) after stimulation of the other branch (the transneuroma response) is shown in E and at a higher oscilloscope gain in F. Gextracellular unit responses recorded from the neuroma head aher stimulation of one branch (first arrow) and another branch (second arrow). H-after crush at the neck (dashed line in A), responses from the head (upper trace) and the other branch (lower trace) were obliterated. Irecordings obtained from site 1 had a conspicuous loss of late activity after the crush. The highergain traces in J show the late activity before (arrow) and after crush. The voltage calibration in B pertains to B-E, and H-J; those of F and G refer to those traces. The time calibration in I is for all traces.
Transneuroma responses could be obtained from either proximal nerve division after stimulation of the other division and were found in 34 preparations with postligature times ranging from 3 weeks to 13 months. In addition to the demonstration of transneuroma activity with compound action potentials, some extracellular unit correlates of this phenomenon were obtained with microelectrode penetrations into the neuroma and proximal nerve divisions. Figure 2G shows extracellularly recorded spikes of a fiber in the head of the neuroma after stimulation of either proximal nerve branch. It was interesting to note the large latency difference for these responses despite the equality of conduction distances measured macroscopically to stimulation sites in the proximal nerves.
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Eight nerve segments from which transneuroma responses had been recorded were used for HRP study. These experiments were carried out to look for axon continuity between proximal nerve divisions and are represented by the schematic of Fig. 3A. The enzyme was applied to the cut end of one proximal division (D-A) which was previously trimmed to a length (X) of 2.8 to 5.0 mm. The nondivided distal portion of the neuroma had a length (Y) of 1.5 to 5.0 mm (1.5 to 2.2 mm in four samples). Because retrograde looping was anticipated to begin at the distal end of the neuroma, it was expected that labeled axons would have to be followed over a distance of X + 2Y. This distance ranged from 7.0 to 12.8 mm (mean 9.9 mm). In the HRP-dipped nerve division, the reaction product inside fine-, medium-, and large-diameter axons had a Go&like appearance comparable to that known for central nervous system axons to follow “injury uptake” of
FIG. 3. HRP-labeled axons in neuroma after application of enzyme to a portion of the proximal nerve. A-schematic representation of preparation and resultant distribution of labeled axons (see text). B-D--50~pm-thick sections from one preparation. In B, labeled axons entered the neuroma from the soaked proximal nerve division but did not spread throughout the neuroma and did not enter nonsoaked proximal nerve. Retrograde turns of tibers at arrows are enlarged in D. In C, many fibers that reach the distal end of the neuroma (another section) turned and continued a retrograde course among labeled antergrade segments. In D, two fibers made sharp retrograde turns. Other reaction product was in more faintly labeled axons as well as in short segments of blood vessels.Magnifications are X25 in B, X63 in C and X 100 in D.
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HRP. Labeled axons were scattered throughout this division and extended into the distal, nondivided stump, but they were always more numerous in the former area than in the latter (Figs. 3B, 4A). In every preparation there was a well defined and cohesive area of the neuroma that contained virtually no labeled axons (stippled area NHB in Fig. 3A; also Fig. 3B). The fibers in this area of the neuroma appeared to be distal extensions of fibers from the proximal nerve division that was not exposed to HRP (D-B). Reaction product
nc. 4. HRP-labeled axons in a whole-mount preparation. A-labeled axons extended from the proximal nerve segment that is twisted diagonally at the upper left. B-enlarged portion of the neuroma showing groups of fibers near the surface of the bundle that made retrograde turns. Magnifications are x47 in A and X150 in B.
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was never found in axons of segment D-B, nor were any examples found of labeled processes directing their course toward area NH-B in a manner suggesting that they would have continued a course into segment D-B. Because the preceeding observations were made in seven whole-mount samples, it was important to consider that reaction product was not formed throughout the tissue mass. This was determined in control nerves that were sectioned after DAB processing as whole segments. These controls revealed densely labeled fibers near the periphery of the bundle, gradual lessening of reaction product density in fibers situated more centrally in the nerve, and absence of reaction product in fibers at distances greater than 200 to 250 pm from the surface. It must be concluded that DAB was not able to react with all HRP present in the whole-nerve samples. However, the pattern of HRP labeling described above for whole-nerve experimental material was also obtained in each of eight 50-pm-thick sections from one neuroma sample (frozen sections taken before reaction), and in this case the probability of undetected HRP was greatly reduced. Among all labeled fibers that coursed within the neuroma, relatively few axons erratically changed direction during any portion of their observed path. Most fibers followed courses that were parallel to the longitudinal axis of the preparation. Frequently the reaction product in a fiber faded gradually and became undetectable, sometimes ending at the distal tip of the neuroma but more often disappearing at intermediate levels of the neuroma. Other labeled fibers made single loops (Figs. 3C, D, 4B), often with an extremely small radius, and continued a retrograde course parallel to and at times slowly rotating around the anterograde segment (Fig. 3D). Such loops were more prominent at the distal end of the neuroma but they could be found at more proximal levels. Except in one preparation (with Y length of 1.5 mm) retrogradely directed segments from loops in the distal extreme of the neuroma could not be readily traced to the level of the nerve division. However, there was little tendency for fibers to direct their retrograde extensions toward the nonlabeled area of the neuroma. Some labeled axons issued collateral branches at distances of 1.5 mm or more from the distal tip of the neuroma. Those collaterals most often took an anterograde direction and generally coursed near the parent axon. DISCUSSION Peripheral nerve fibers that enter a neuroma show a number of abnormal electrophysiologic properties. These include increased mechanosensitivity, spontaneous firing, and afterdischarges (3, 5, 6, 12, 13, 19, 20, 23). Another property attributed to neuroma but not to normal nerve is a high safety factor axonal “cross-talk” (3, 13, 20). This phenomenon was originally proposed to explain experiments where a neuroma existed in sciatic nerve and
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where stimulation of a dorsal root that was cut centrally led to activation of ventral root fibers (20). Although the mechanism of this effect is uncertain, it has been assumed that morphologic alterations within the neuroma such as close apposition of the fine-caliber axonal sprouts could allow for electrical coupling between the axons (2). No contact specializations such as gap junctions or synaptic-like profiles have been described for fibers entering neuroma, and recent studies have raised the prospect that retrograde regeneration from a neuroma may at least partially account for retrograde impulse activity (3). One aim of the present work was to establish whether or not some physiologic properties associated with peripheral neuroma studied in viva could also be shown with a simple in vitro model. It was found that abnormal responses were reliably elicited in the in vitro preparation. The most conspicuous abnormality occurred when stimulation of one arbitrarily formed proximal division of a nerve resulted in transneuroma activity in fibers of another, nonstimulated, division. There are several arguments against the possibility that this activity resulted from inadvertent stimulus currents acting on tissues remote from the stimulation electrodes (i.e., virtual cathode effects). Firstly, the transneuroma response occurred with a relatively long latency whereas electrotonic spread of stimulus current could be expected to produce short-latency responses. Secondly, the transneuroma response was abolished by crush at the neck of the neuroma. And finally, similar responses could not be elicited in simulations of the neuroma experiments with split segments ending as nondivided normal nerve. Although our electrophysiologic data do not entirely establish the morphologic basis for transneuroma responses, several conclusions can be drawn. First, the very occurrence of these responses in the split-neuroma preparation confirms that at least some portion of the transneuroma response must be due either to direct axonal connection (via retrograde regeneration) between proximal nerve divisions or to axonal cross-talk in the head of the neuroma, or to some combination of these factors. However, if a single stimulus gives rise to repetitive firing, parts of the early and late components of the transneuroma response may originate from the same fiber. The late activity in the response of the stimulated branch may share a common mechanism to the transneuroma response, i.e., resulting from retrograde regeneration or cross-talk. There is the additional possibility of impulse reflection back into the stimulated branch due to morphologic nonhomogeneity of the axon region within the neuroma (22). We have not been able to distinguish between these three possibilities from our electrophysiological studies. However, we would like to point out that if axons entering neuroma regenerate in a retrograde manner and loop so that stimulation of a given whole-nerve branch leads to activation of both ends of the looping axon, then under ideal conditions, collision should occur and the late activity as recorded from the
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stimulated branch should be obliterated. Similarly, if cross-talk occurs within the neuroma, collision should also occur after stimulation of a given branch with attendant loss of late activity. However, late activity in the stimulated branch in our experiments was prominent. Yet, the highly nonhomogeneous morphologic organization of the axons as they enter the neuroma could significantly alter the safety factor for transmission and could explain the lack of collision. Specifically, the conduction safety factor could be lower for one branch thereby leading to impulse failure in or near the neuroma. If failure occurs along the propagation path of one arm of the looping axon (or one element of a coupled pair of axons), the other activated axon may (given a high enough safety factor) continue retrograde propagation and give rise to activity in the stimulated branch. A second aim of this work was to show, with HRP diffusion, whether or not axons would course toward the neuroma from one division of the proximal nerve and then proceed into the other proximal division. Our failure to demonstrate this direct connectivity is offered only as tentative morphologic evidence that axonal cross-talk may be the determinant of the transneuroma response. Although no HRP was found in the nonsoaked nerve division of eight samples, the following criticisms can be made: (i) The observed transneuroma responses may have been generated by retrograde activity in a relatively small set of axons. In the majority of eight samples, relatively few retrograde segments could be traced to and beyond the region of nerve division. Thus, it is possible that HRP did not diffuse far enough, in the relevant set of axons, to enter the nonsoaked division. (ii) In seven of eight samples (whole-tissue specimens) as much as 40% of the core tissue volume that contained HRP probably did not form reaction product. Thus a small set of retrograde loops contained within this volume in the nonsoaked division would not have formed reaction product despite possibly containing HRP. (iii) The HRP may not have diffused into all axons of the soaked division and therefore a small set of fibers that ultimately coursed into the nonsoaked division may have been consistently excluded from the set of labeled axons. It should be emphasized that the latter two possibilities would have acted randomly and therefore seem improbable causes of failure to detect a single positive outcome in eight samples. Furthermore, several features of our data should be given appropriate weight: (a) Retrograde extensions were traced to the region of nerve division thereby indicating that diffusion distance of HRP was sufficient in a set of axons. (b) Retrograde extensions originated from loops in all regions of the neuroma (not just from those in the distal termination), further increasing the likelihood of tracing labeled axons into the nondipped branch. (c) In all HRP experiments, retrograde segments rarely
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coursed into any part of the nonlabeled mass of the neuroma. (d) There was a marked tendency for a fiber’s retrograde segment to course very close to its anterograde segment, suggesting that both segments would most likely be found in the same proximal division of the nerve. Taken together, these observations lend support to the hypothesis that retrograde transneuroma activity in these experiments may have been caused by cross-talk rather than by direct continuity of axons between the divisions of the proximal nerve. Other investigators have suggested that “ephaptic” (electrical field effect) interactions account for this activity (2, 3, 5, 13, 20, 23). However, there is no direct confirmation of this proposal. Several morphologic features of axons entering neuroma formation offer support for this proposal. The fibers that enter neuroma give rise to fine-caliber axonal sprouts that can become closely apposed with few intervening glial elements (2). The reasoning to explain why densely packed axonal sprouts are more likely to give rise to electrical interactions is that the extracellular resistance is increased from the tight axon packing and more current will be shunted across neighboring axon membranes ( l&20). As few glial elements intervene, there are few alternative current pathways except through the extracellular space and the neighboring axons. In the cerebellar cortex a similar morphologic organization is present, i.e., dense, fine-caliber axon packing with few intervening glial elements. Activity-dependent changes in extracellular potassium can lead to profound changes in axonal excitability of nonactivated nonmyelinated axons (10). Whereas the excitability changes brought about from change in the extracellular ionic environment can last for seconds, cross-talk from electrical field effects more closely approximates the time course of the action potential (9). As pointed out by Seltzer and Devor (20), the high safety coupling between nerve fibers in neuroma is not likely due to increase in extracellular potassium because its time course is brief and the effect is nonaccumulative with repetitive stimulation. Analytical studies of interactions of nonmyelinated fibers (4, 14) and arguments set forth by Rasminsky (18) indicate that myelinated fibers may be more susceptible than nonmyelinated axons to electrical field effect excitation from surround impulse activity. This may be because myelinated fibers have a relatively low membrane capacitance and low axial resistance, both of which lead to an increased probability of interaction. Indeed, Rasminsky (18) demonstrated in dysmyelinated fibers of the dystropic mouse that interactions more readily proceed from unmyelinated to myelinated axons. Previous studies on axonal interactions or cross-talk in neuroma have been carried out in Go. In this study we demonstrated that nerves terminating
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in neuroma can be maintained in vitro and that a transneuroma response can be demonstrated in this relatively simple in vitro preparation. An advantage of this preparation is that alteration in the ionic environment and direct application of drugs to the neuroma can be made. The technique should prove valuable in future studies to determine in more detail the specific mechanisms responsible for transneuroma activity and for other abnormal activity of pathological fibers entering the neuroma. REFERENCES 1. ARVANITAKI, A. 1942. Effects evoked in an axon by activity of a contiguous one. J. Neurophysiol. 5: 89-108. 2. BERNSTEIN, J. J.. AND D. PAGNANELLI. 1982. Long-term axonal apposition in rat sciatic nerve neuroma. J. Neurosurg. 57: 682-684. 3. BLUMBERG, H., AND W. JANIG. 1982. Activation of fibers via experimentally produced stump neuromas of skin nerves: ephaptic transmission or retrograde sprouting. Exp. Neural.
16: 468-482.
4. CLARK, J. W., AND R. PLONSEY. 1970. A mathematical study of nerve fiber interaction. Biophys.
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5. DEVOR, M., AND J. J. BERNSTEIN. 1982. Abnormal impulse generation in neuromas: electrophysiology and ultrastructure. Pages 363-80 in J. OCHOA AND B. CULP, eds., Abnormal Nerves and Muscles as Impulse Generators. Oxford Univ. Press, London/New York. 6. GROVRIN-LIPPMANN, R., AND M. DEVOR. 1978. Ongoing activity in severed nerves: source and variation with time. Brain Res. 159: 406-410. 7. HUIZAR, P., M. KUNO, AND Y. MIYATA. 1975. Electrophysiological properties of spinal motoneurones of normal and dystrophic mice. J. Physiol. (London) 248: 231-246. 8. KATZ, B., AND 0. H. SCHMITT. 1942. A note on interaction between nerve fibers. J. Physiol. (London) 100: 369-37 1. 9. KOCSIS, J. D., J. A. RUIZ, AND K. CUMMINS. 1982. Modulation of axonal excitability mediated by surround electrical activity; an intra-axonal study. Exp. Brain Res. 47: 15 l153. 10. Kccsn, J. D., R. C. MALENKA, AND S. G. WAXMAN. 1983. Effectsof extracellular potassium on the excitability of the parallel fibers of the cerebellar cortex. J. Physiol. (London) 334: 225-244.
1I. KOCSIS,J. D., J. A. RUIZ, AND S. G. WAXMAN. 1983. Maturation of mammalian myelinated fibers: changes in action potential characterization following 4-aminopyridine application. J. Neurophysiol.
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12. KORENMAN, E. M. D., AND M. DEVOR. 198 I. Ectopic adrenergic sensitivity in damaged peripheral nerve axons in the rat. Exp. Neural. 72: 63-81. 13. LISNEY, S. J. W., AND C. M. POVER. 1983. Coupling between fibres involved in sensory nerve neuroma in cats. J. Neural. Sci. 59: 255-264. 14. MAEDA, K.. T. YAGI, AND A. NOCXJCHI. 1980. Induced excitation and synchronization of nerve impulses in two parallel unmyelinated fibers. IEEE Trans. Biomed. Eng. 27: 139145. 15. MARRAZZI, A. S., AND R. LORENTE DE N6. 1944. Interaction of neighboring fibers in myelinated nerve. J. Neurophysiol. 7: 83-101. 16. PRESTON,R. J., J. D. KOCSIS, AND S. G. WAXMAN. 1983. Axon-myelin anomolies revealed
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17. 18. 19. 20. 21. 22. 23.
KOCSIS, PRESTON, AND TARG by diaminobenzidine reaction with intra-axonal horseradish peroxidase. Sot. Neurosci. Abstr. 9: 50. kUdN Y CAJAL, S. 1928. Degeneration and Regeneration of the Nervous System. Vol. 1. Oxford Univ. Press, London. RASMINSKY, M. 1980. Ephaptic transmission between single nerve fibres in the spinal nerve roots of dystrophic mice. J. Physior (London) 305: 15 l- 169. SCADDING,J. W. 198 1. Development of ongoing activity, mechanosensitivity, and adrenalin sensitivity in severed peripheral axons. Exp. Neural. 73: 345-364. SELTZER,Z., AND M. DEVOR. 1979. Ephaptic transmission in chronically damaged peripheral nerves. Neurology 29: 106 I-1064. SUNDERLAND, A. 1968. Nerves and Nerve Injuries. Livingston, Edinburgh. SWADLOW,H. A., J. D. Kocs~s, AND S. G. WAXMAN. 1980. Modulation of impulse conduction along the axonal tree. Annu. Rev. Biophys. Bioeng. 9: 143-79. WALL, P., AND M. GUTNICK. 1974. Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Exp. Neural. 43: 580-593.